intracellular ace2  (Sino Biological)


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    Name:
    ACE2 Angiotensin Converting Enzyme 2 Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant Human ACE2 Angiotensin Converting Enzyme 2 rh ACE2 Angiotensin Converting Enzyme 2 NP 068576 1 1M 740S ACE2 Angiotensin Converting Enzyme 2 specific IgG was purified by Human ACE2 Angiotensin Converting Enzyme 2 affinity chromatography
    Catalog Number:
    10108-T56
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    Human
    Applications:
    WB,ELISA,IHC-P
    Immunogen:
    Recombinant Human ACE2 / Angiotensin-Converting Enzyme 2 Protein
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological intracellular ace2
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of <t>ACE2‐overexpressing</t> cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Produced in rabbits immunized with purified recombinant Human ACE2 Angiotensin Converting Enzyme 2 rh ACE2 Angiotensin Converting Enzyme 2 NP 068576 1 1M 740S ACE2 Angiotensin Converting Enzyme 2 specific IgG was purified by Human ACE2 Angiotensin Converting Enzyme 2 affinity chromatography
    https://www.bioz.com/result/intracellular ace2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    intracellular ace2 - by Bioz Stars, 2021-06
    95/100 stars

    Images

    1) Product Images from "Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors"

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    Journal: Small Methods

    doi: 10.1002/smtd.202001031

    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Figure Legend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Techniques Used: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay

    Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p
    Figure Legend Snippet: Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Techniques Used: Infection, Incubation, Confocal Microscopy, Fluorescence

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    Produced:

    Article Title: Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies
    Article Snippet: The L452R mutation is the predominant mutation found in the recently described variants CAL.20A, derived from clade 20A (lineage B.1.232,) and CAL.20C, derived from clade 20C (lineage B.1.429), that are associated with multiple outbreaks in California. ( - ). .. SARS-2 RBD variants with single mutations were produced and their affinity for human ACE2 and the three VH H-huFc antibodies was determined by BLI ( S6-9 Fig. and S6 Table ). .. In all cases, individual mutations were shown to have little to no impact on ACE2 binding, suggesting minimal impact on viral infection.

    Infection:

    Article Title: Collapsing Glomerulopathy in a Patient With Coronavirus Disease 2019 (COVID-19)
    Article Snippet: Discussion Possible mechanisms for kidney injury in COVID-19 include direct infection of the kidney as well as cytokine storm related to sepsis. .. Direct infection of the renal parenchyma is possible because the renal proximal tubule cells highly express angiotensin-converting enzyme 2, the cellular entry receptor for the SARS-CoV-2 virus., The virus likely gains access to the kidney through the bloodstream, as approximately 15% of patients were found to have RNAemia in one series. .. Cytokine storm has been described previously both in animal models and humans infected with other highly pathogenic human coronaviruses, including severe acute respiratory syndrome and Middle East respiratory syndrome., , Evidence of cytokine storm has also been documented in patients with COVID-19., .

    Blocking Assay:

    Article Title: RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response
    Article Snippet: RBD-Fc protein (2 µg/ml) was coated on the ELISA plates at 4 °C overnight. .. Then the plates were blocked using blocking buffer (PBST containing 5% BSA) at 37 °C for 2 h. A series of concentrations of human ACE2 protein was added to the plate and incubated at 37 °C for 2 h. After 4 times of washing using PBST, rabbit anti-human ACE2-specific antibody (Sino Biological, Beijing, China) was added to the plate and incubated for 2 h at 37 °C. .. Subsequently, HRP-conjugated goat anti-rabbit IgG (Dako, Denmark) was added to the plates and incubated for 1 h at 37 °C.

    Incubation:

    Article Title: RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response
    Article Snippet: RBD-Fc protein (2 µg/ml) was coated on the ELISA plates at 4 °C overnight. .. Then the plates were blocked using blocking buffer (PBST containing 5% BSA) at 37 °C for 2 h. A series of concentrations of human ACE2 protein was added to the plate and incubated at 37 °C for 2 h. After 4 times of washing using PBST, rabbit anti-human ACE2-specific antibody (Sino Biological, Beijing, China) was added to the plate and incubated for 2 h at 37 °C. .. Subsequently, HRP-conjugated goat anti-rabbit IgG (Dako, Denmark) was added to the plates and incubated for 1 h at 37 °C.

    Article Title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells
    Article Snippet: Then, cells were fixed with PFA 2% in PBS for 15 min, rinsed three times with PBS, permeabilized for 15 min with Triton-X 100 (Sigma) 0.1% in PBS for 15 min, and rinsed four times with PBS + 0.5% BSA (PBB). .. Fixed cells were incubated overnight at 4 C° with 200 μl of a solution of anti-ACE2 IgG, and either Spike RBD-mFC Recombinant Protein (40592-V05H-100, SinoBiological), or anti-caveolin-1 IgG, or anti-CD71 IgG. .. After rinsing four times with PBB, immunolabeled cells and negative controls were incubated for 1h with a solution of 2 fluorescent-labeled secondary antibodies in PBB (see secondary antibody section in Materials and Methods) and then rinsed four times with PBB and three times with PBS.

    Article Title: Evidence for Gastrointestinal Infection of SARS-CoV-2
    Article Snippet: Samples were embedded with paraffin and then stained with H & E. For immunofluorescent staining, 3-μm-thick sections were dewaxed in xylene, rehydrated in alcohol, and washed in distilled water 3 times before microwave repair. .. After washing 3 times in phosphate-buffered saline with Tween (PBST), sections were incubated with 10% goat serum in PBST for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies (anti-ACE2, Sino Biological, Beijing, China, 10108-T56, 1:500; anti-nucleoprotein, Sino Biological, 40143-T62, 1:500). .. The slides were incubated with secondary antibodies (Alexa Fluor 647–conjugated goat anti-rabbit IgG, bs-0296G-AF647, 1:100; Bioss, London, UK) for 1 hour at room temperature followed by washing 3 times with PBST.

    Article Title: Rapid Development of SARS-CoV-2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates
    Article Snippet: The plates were then incubated with sequentially 1:5 diluted ACE2 starting from 50 μg/mL in ELISA blocking buffer. .. After 1 h of incubation, the plates were washed with PBS-T five times, and 100 μL of hACE2-specific rabbit antibody (Sino Biological Inc.) diluted at a ratio of 1:5000 was added to the wells and incubated at 37 °C for 1 h. The plates were washed with PBS-T five times, and a 1:5000 dilution of Horseradish Peroxidas (HRP)-conjugated goat antirabbit IgG antibody (Promega) was added and incubated for 45 min at 37 °C. .. After the final round of washing to remove all unspecifically bound antibodies, the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Sangon Biotech) was added to initiate the chromogenic reaction.

    Recombinant:

    Article Title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells
    Article Snippet: Then, cells were fixed with PFA 2% in PBS for 15 min, rinsed three times with PBS, permeabilized for 15 min with Triton-X 100 (Sigma) 0.1% in PBS for 15 min, and rinsed four times with PBS + 0.5% BSA (PBB). .. Fixed cells were incubated overnight at 4 C° with 200 μl of a solution of anti-ACE2 IgG, and either Spike RBD-mFC Recombinant Protein (40592-V05H-100, SinoBiological), or anti-caveolin-1 IgG, or anti-CD71 IgG. .. After rinsing four times with PBB, immunolabeled cells and negative controls were incubated for 1h with a solution of 2 fluorescent-labeled secondary antibodies in PBB (see secondary antibody section in Materials and Methods) and then rinsed four times with PBB and three times with PBS.

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    Sino Biological intracellular ace2
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of <t>ACE2‐overexpressing</t> cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Intracellular Ace2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/intracellular ace2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    intracellular ace2 - by Bioz Stars, 2021-06
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    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Article Snippet: [ ]Commercial antibodies were used to detect intracellular ACE2 (Sino Biological, 10108‐T56) and GAPDH (Proteintech, HRP‐60004) according to the manufacturer's instructions.

    Techniques: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay

    Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Article Snippet: [ ]Commercial antibodies were used to detect intracellular ACE2 (Sino Biological, 10108‐T56) and GAPDH (Proteintech, HRP‐60004) according to the manufacturer's instructions.

    Techniques: Infection, Incubation, Confocal Microscopy, Fluorescence